The research mission of the Inorganic Toxicology (IT) Group is to characterize responses to carcinogenic inorganics to elucidate mechanisms under a single project titled Mechanisms of Inorganic Toxicology. The major focus is on arsenic with a smaller Cd project. Inorganic carcinogens are major human health hazards and defining mechanisms is key to defining risk. The development of rodent cancer models for arsenic is a recent advent in which the IT Group members played a major role. We also extensively use target relevant cellular models. Cd is a well defined human and rodent carcinogen, so cell models are used to define mechanisms in established/suspected human targets. Accepted human targets for inorganic arsenic are the lung, skin and urinary bladder and suspected targets are the prostate, liver and kidney. Evidence linking arsenic exposure to human kidney cancer was recently augmented. Cd clearly targets the human lung but the prostate and kidney are considered likely targets. Inorganic arsenic is carcinogenic after ingestion or inhalation and cancer most commonly occurs after environmental exposure. Tens of millions of people worldwide are exposed to unhealthy arsenic levels in drinking water but questions remain about health issues of low level exposures, making elucidation of mechanisms all the more important. With Cd environmental exposure may play an important role in cancer. Members of IT Group developed a mouse transplacental cancer model in which multiple studies show arsenic exposure in utero causes or facilitates tumors in adulthood at various sites, including known human targets such as the lung, liver, skin and urinary bladder. These studies were designed and performed entirely within the NCI, and do not reflect the standard procedures, depth or extent of a typical NTP tumor endpoint study. None-the-less these mouse studies actually stimulated work on early life human arsenic exposure from the drinking water and its association with adulthood cancers in humans. Human data now link early life drinking water arsenic exposure and lung, liver and kidney cancer. The population studied comes from Chile and was exposed to high natural levels of drinking water arsenic from 1958 until it was remediated in 1970. The results of these studies are so stunning it prompted Dr. Allen Smith, a prominent University of California at Berkeley epidemiologist to say that this arsenic exposure in the drinking water had resulted in the greatest increases in cancer mortality in adults ever associated with early-life environmental exposure. There were also high dose pulse exposures in humans in early life that caused cancer in adulthood. In this case inorganic arsenic- contaminated powdered milk caused a mass poisoning event of infants in the mid-1950's in Japan and is now linked to cancers in the survivors, including liver cancer. In our mouse studies we now have also lowered the dose required for carcinogenesis by more than 10-fold using whole life exposure, which more reasonably duplicates typical human environmental exposure. It is clear from the literature that exposure to even lower doses of inorganic arsenic in gestation in mice induces molecular changes after birth consistent with the possibility of cancer development in target tissues, like lung, though no actual tumor data exist to fortify this suspicion. Both human and rodent evidence indicate early life is a time of sensitivity to inorganic arsenic exposure. The early life period, including in utero and neonatal life, is also a time of high stem/progenitor cell activity due to organogenesis, global proliferative growth, etc. Since inorganic arsenic as a cancer chemotherapeutic is known to impact stem cell (SC) programming as part of its therapeutic mode of action, this lead us to hypothesize early on that in early life SCs could be a key target population of arsenic carcinogenesis. Perinatal arsenic exposure, which induces or predisposes to mice lung, skin, urinary bladder, liver or kidney tumors as adults, also causes an over-abundance putative cancer SCs (CSCs) in many of these same tumors. We also find superior innate and acquired arsenic resistance in human and rodent SC lines, involving general and arsenic-specific adaptation. Malignant transformation of a heterogenous mature prostate line with arsenic causes a stunning putative CSC overproduction. A major issue is how arsenic can specifically target SCs and what the molecular manifestations of this targeting are. We have specific, target relevant SC cell lines which are available to use to look into these important questions. Recent human data indicate where elevated arsenic exposure is remediated, despite long-term exposure cessation, cancer risk remains elevated in lung and bladder for at least 40 years. This fortifies the notion that a quiescent, long-lived cell (i.e. SCs) passes damage along for years. With chronic arsenic exposure, cells adapt in various ways such as via altered methyl metabolism, oxidant stress response or enhanced export. For instance, arsenic-transformed skin keratinocytes adapt via diminished oxidative stress response. Once adapted, cells are cross-adapted to ultraviolet (UV) irradiation, but still show UV-induced oxidative DNA damage (ODD) at a level higher than control due to apoptotic by-pass, likely a basis of skin co-carcinogenesis with arsenic plus UV. Inorganic arsenic undergoes enzymatic biomethylation (BML) by a specific methyltransferase (AS3MT) and S-adenosyl-methionine (SAM) as the cofactor. Arsenic BML was early on thought to be adaptive but many target cells of arsenic carcinogenesis do not BML arsenic. The role of arsenic BML and ODD generation and malignant transformation has been tested in multiple models. When a BML-capable liver line and a BML-deficient prostate line are exposed through transformation, ODD occurred in BML-capable cells prior to transformation but BML-deficient cells showed no ODD despite exposure past transformation. This indicates multiple mechanisms. Direct exposure to the BML product, methylarsonous acid (MMA3+) indicates that both BML-capable and BML-deficient cells show similar patterns and levels of ODD which is very illuminating with regards to early steps in methylation and oxidative stress. Thus, arsenic BML is obligatory for ODD, and hastens acquired cancer phenotype, but cells can acquire a cancer phenotype without ODD, again implicating multiple mechanisms. Furthermore MMA3+ needs no further methylation to produce ODD. This may be important for the lung, as a genetic predisposion to poorly methylate arsenic past MMA was recently linked to lung cancer in humans. This could indicate a unique sensitivity to MMA3+ in lung cells. The prostate is a potential human target of Cd. In contrast to arsenic, which selects for SC accumulation, Cd early on selectively kills SCs. Cd caused 95% cytolethality in our prostate SC line exposed to a non-toxic, but transforming, level for the heterogeneous parental epithelial ine. Though depleted, remaining SCs rapidly re-emerge and undergo transformation. We are determining if Cd has transformed these SCs and observing these SCs and observing the mature cell line for selection of hyper-resistant SCs. The pancreas is another potential human Cd target. Human pancreatic epithelial cells are transformed by Cd, and form aggressive CSC-like cells, though not in excess. We have developed a human lung epithelial cell transformant with Cd from a line that would produce adenocarcinoma, the tumors linked to Cd in humans and rodents. Cd appears to work, at least in part, by epigenetic mechanisms, including modification of DNA methylation.